PROJECT SUMMARY/ABSTRACT A GGGGCC hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of frontotemporal dementia (FTD) and familial amyotrophic lateral sclerosis (ALS), and the mutation is frequently seen in apparent sporadic patients. The underlying mechanisms by which the C9ORF72 HRE causes ?c9FTD/ALS? are poorly defined. Multiple studies, including our own, support a gain-of-function mechanism of neurotoxicity mediated by the HRE. Expanded repeats may generate toxic RNAs that sequester RNA-binding proteins. They may also be translated via repeat-associated non-ATG (RAN) translation into toxic ?c9RAN proteins? of repeating dipeptides. Furthermore, multiple studies from our lab and others suggest that disruption of the nuclear pore and/or nucleocytoplasmic transport is a primary cause of neurodegeneration in yeast, fly, and induced pluripotent stem (iPS) cell models of c9FTD/ALS. In addition, our recent use of c9FTD/ALS iPS cell-derived neurons (iPSN) suggests they are a valuable and highly relevant preclinical model to uncover candidate therapies. This proposal will investigate the mechanism by which the C9ORF72 HRE disrupts nucleocytoplasmic transport and nuclear pores utilizing complementary models, which will include c9FTD/ALS iPSN and patient brain tissues, as well as brain tissue from (G4C2)66 mice (generated by Project 2), an in vivo model that recapitulates behavioral and neuropathological features of c9FTD/ALS. Our data suggest the defects in nucleocytoplasmic transport are fundamental events coupled with neurodegeneration in c9FTD/ALS. The proposed studies, first in iPSN and then validated in vivo with Project 2, will establish whether candidate therapies emerging from Project 1 are able to mitigate such detrimental defects. First, we will comprehensively investigate nuclear pore complex (NPC) function and pathology in c9FTD/ALS iPSN and rodent models. These experiments will define the specific role of NPC components in c9FTD/ALS pathophysiology and highlight targets worthiest of experimental manipulation or monitoring for molecular efficacy. Findings from these studies will guide our efforts to identify therapeutic strategies that mitigate nuclear pore dysfunction and pathology in c9FTD/ALS iPSN and rodent models. Such strategies will include genetic expression of selected nucleoporins or other mediators of nucleocytoplasmic transport (e.g. RanGAP1), and r(G4C2)exp-binding small molecules from Project 1. Finally, we will identify therapeutic strategies that rescue the heightened susceptibility of c9FTD/ALS iPSN to cellular stressors. C9ORF72 HRE toxicity may occur via disruption of NPC function and other events distal to the mutation that impair cellular function. These studies will provide a fundamentally important general readout of the therapeutic benefit of blocking HRE toxicity using r(G4C2)exp-binding small molecules or manipulating other mechanistic targets identified as part of our proposed studies.